S. aureus is a major human pathogen; because of emerging antibiotic resistance, there is a need to develop new antimicrobial strategies. We have identified a regulatory system involved in autolysis as a novel target. The integrity of the cell wall in bacteria is generally maintained by two competing processes: cell wall synthesis and autolytic activity. While synthesis of the cell wall synthesis is mediated by PBP, murein hydrolases mediate the lytic process. An imbalance between these two processes may lead to cell lysis. We recently identified a regulatory locus called rat in S. aureus; a mutation in rat resulted in a growth defect and increased murein hydrolase activity while leaving cell wall synthesis relatively unperturbed. Consequently, the rat mutant had enhanced detergent and penicillin induced lysis as compared with the parental strain. As the Rat protein is more homologous to the MarR protein family, we hypothesize that the rat gene product is a unique regulator of autolytic activity in S. aureus. Rat also represses another gene called sarV, a member of the recently described SarA protein family. Contrary to rat, a mutation in sarV rendered the mutant more resistant to lysis. We speculate that rat may repress sarV to control autolysis in S. aureus. To understand the inner working of rat and sarV and to facilitate the development of a novel therapeutic approach, we propose the following five specific aims: I) characterizing the interaction and regulatory pathways of rat and sarV in autolysis in vitro; II) Evaluating the role of rat and sarV in detergent and antibiotic mediated cell lysis; III) Determining the role of rat and sarV in the regulation of virulence determinants in vitro; IV) Assessing the target genes of rat and sarV by determining the transcriptional profile and target autolysins of the rat mutant and strains expressing a high level of sarV and determining the gene promoters to which Rat and SarV bind; IV) Evaluating the role of rat and sarV in virulence and responses to antibiotic therapy in the rabbit endocarditis model. Upon completion of these studies, we hope to understand the role of rat and sarV within the network of regulators that control autolysis. Our long-term goal is to promote autolysis with an anti-Rat or pro-SarV strategy without developing an untoward impact on virulence.